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. 2021 Aug 4;7(32):eabe9254. doi: 10.1126/sciadv.abe9254

Fig. 9. Ubiquitinated GRB2 releases MRE11 at the DSB sites.

Fig. 9

(A) HEK293T cells expressing Strep-GRB2 were treated with 5-Gy IR, lysed immediately (0), or allowed to recover for the designated time. Strep-tagged GRB2 was precipitated and immunoblotted with MRE11 antibody. Input controls are shown in the bottom three panels. (B) Control or RBBP6-KD1 HEK293T cells overexpressing Strep-GRB2 were treated with 5-Gy IR, lysed immediately, or allowed to recover for the designated time. Strep-GRB2 was precipitated from total extract and immunoblotted with indicated antibodies along with input controls. (C) Strep-tagged WTGRB2 or K109AGRB2 mutant precipitated from 5-Gy IR–treated (0) or post-recovery (2 hours) and analyzed with indicated antibodies. (D) DR-GFP reporter assay for control (Ctrl) and two RBBP6-KD U2OS cells. (E) DR-GFP reporter assay for control, GRB2-KO (KO), and K109A reconstituted (KO + K109A) U2OS cells. The significance was analyzed by two-sided Student’s t test. **P ≤ 0.01 and ***P ≤ 0.001. (F) Schematic proposed model depicting the role of GRB2 in the DSB repair. IR creates DSBs and phosphorylation of H2AX, which serves as a docking site for the recruitment of the GM complex. Ubiquitination of GRB2 at K109 by RBBP6 releases GRB2 from MRE11, while deubiquitination of GRB2 possibly by PSMD14 enables GM reassociation.